We propose to continue and extend ongoing studies of the mechanistic enzymology and molecular genetics of terpenoid biosynthesis. The focus will be on a family of sesquiterpene synthases which catalyze the cyclization of the universal acyclic precursor farnesyl diphosphate (FPP, 1) to pentalenene (2), aristolochene (3), germacradienol (4), and amorphadiene (5), as well as the metabolic conversion of these sesquiterpenes to antibiotics such as the antibiotic pentalenolactone (6) or to geosmin (7), the characteristic oderiferous constituent of Streptomyces species. Three broad and closely interrelated questions must be addressed: 1) How does a cyclase impose a specific folding pattern or conformation on its acyclic substrate FPP and derived intermediates? 2) How does a cyclase manage positively charged intermediates, including catalysis of the initial ionization of the substrate, through stabilization of cationic intermediates, to termination of the reaction by quenching of positive charge? and 3) What is the nature of the cyclase active site? To answer these questions and to explore terpenoid mechanism and structure space as broadly as possible, we have devised a set of mutually complementary experimental approaches involving : A. Determination of the X-ray crystallographic structures of wild-type and mutant terpenoid synthases, both substrate-free and with bound substrate and intermediate analogs, in collaboration with Prof. David W. Christianson (University of Pennsylvania); B. Isolation, expression and mechanistic investigation of new terpenoid synthases from bacterial and fungal sources based on genomic sequence information; C. Site-directed mutagenesis of terpene synthases, exploiting the tendency of such mutants to produce mixtures of aberrant products that are diagnostic of the normally cryptic intermediates of the carbocationic cyclization cascade; D. Mechanistic studies of terpene synthases, using isotopic labeling, isotopically sensitive branching experiments, and pre-steady state, rapid chemical quench kinetics to define the mechanism of formation of individual sesquiterpenes from FPP; E. Heterologous production of cyclic sesquiterpenes and terpenoid metabolites in E. coli. In collaboration with Prof. Jay D. Keasling (University of California, Berkeley), we will use an engineered strain of Escherichia coli to express individual sesquiterpene synthases as well as entire terpenoid biosynthetic gene clusters. This experimental system will be exploited to produce 10-100 mg quantities of terpenoid metabolites for structural characterization and to define the complete set of genes, enzymes, and metabolic intermediates in terpenoid biosynthetic pathways.